Inactivation Of Urease Research Articles

Immiscible organic solvent inactivation of urease, chymotrypsin, lipase, and ribonuclease: Separation of dissolved solvent and interfacial effects

A new technique with controlled interface generation allows separation and quantitation of enzyme inactivation by both solvent/aqueous interface and dissolved solvent. This has now been used in n-butanol, isopropylether, 2-octanone, n-hexane, n-butylbenzene, and n-tridecane. Ribonuclease was stable with all the solvent/aqueous interfaces studied. Chymotrypsin was mainly inactivated by the more hydrophobic solvent/aqueous interfaces, whereas lipase was only inactivated by the less hydrophobic solvent/aqueous interfaces. Urease was inactivated by some interfaces, but not all, without an obvious trend. Thus, the commonly expected simple relationship with solvent polarity (e.g., log P) does not apply when interfacial inactivation is determined specifically. Greater dissolved solvent inactivation occurred with the more polar solvents, though only a general trend was apparent with log P. A better correlation was noted with the Hilde-brand solubility parameter. Interfacial effects are discussed with reference to enzyme molecular weight, denaturation temperature, hydrophobicity, and adiabatic compressibility.

Diethylpyrocarbonate reactivity of Klebsiella aerogenes urease: effect of pH and active site ligands on the rate of inactivation.

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.

Monoclonal antibodies against urease from Canavalia ensiformis

Monoclonal antibodies against purified urease (EC 3.5.1.5) from Canavalia ensiformis were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. All culture wells exhibited hybrid growth and 25% of these ( ie 45 culture wells) contained anti-urease activity. Two positive hybrid cells were cloned twice by the limiting dilution method and three hybridoma clones (B6F, C4F and B18) secreting monoclonal antibodies were selected ar random for purification and characterisation purposes. All three cell lines secreted monoclonal antibodies of IgM class which were purified by gel filtration chromatography on Sephacryl S-200 column with a final recovery of 85% and a purification factor of about 18. The purified preparations were apparently homogenous on native PAGE running with a M r of 920 000 Da. mAbs were highly specific for jack bean urease as determined by Western blotting. The affinity constants (K) for these mAbs ranged fro 10 8 to 10 9 1 mol −1. mAb B6F inhibited about 65% of urease activity whereas C4F and B18 stimulated the enzyme activity slightly by 20%. The presence of 2-mercaptoethanol in incubation mixtures protected urease from inactivation by B6F. Urease inactivation by B6F could be reversed by addition of 2-mercaptoethanol which reactivated most of the partially inactive enzyme. Gel filtration chromatography of purified urease exhibited two protein peaks with M r values of 290 000 and 90 000 Da which revealed antibody activity. This result suggests that the mAb B6F recognizes the trimeric as well as the monomeric forms of urease.

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.

Urease immobilized on chitosan membrane. Inactivation by heavy metal ions

AbstractHeavy metal ion inactivation of urease covalently immobilized on glutaraldehyde‐pretreated chitosan membrane was compared with that of free urease. The relative toxicity sequence of metal ions toward free urease: Hg2+ > Ag+ > Cu2+ > Ni2+ > Cd2+ > Zn2+ > Co2+ > Fe2+ > Pb2+ > Mn2+ correlates well with metal sulfide solubility product constants (except for Fe3+). The stability of urease against metal ion inactivation was considerably improved after immobilization: Ni2+, Co2+, Fe3+, Pb2+ and Mn2+ do not inactivate the immobilized urease in the range of concentrations at which free urease is thoroughly inactivated. Hg2+, Ag+, Cu2+, Zn2+ and Cd2+ inactivate the enzyme at considerably higher concentrations. The improved stability may be a consequence of structural changes introduced to the enzyme by the immobilization and of chelating effect of chitosan.

Inactivation of trypsin inhibitor, lectin and urease in soybean by hydrothermal treatment

AbstractThe effects of hydrothermal treating parameters on trypsin inhibitor (TI), lectin and urease activities in whole soybean seeds were investigated by Response Surface Analysis (RSA). Independent variables with equidistant levels examined in this study were the following: treating temperature (levels: 70, 85 and 100°C), treating time (levels: 10, 30 and 50 min), and soaking time (2, 4 and 6 h). The functions between treating parameters and responses values of TI, lectin and urease activities in treated soybean were calculated by multiple, non‐linear regression analysis and analysis of variance.Mathematical models were developed in this study to predict TI, lectin and urease activities in soybean during hydrothermal processing and they have been found to be significant (P[%] = 0.1, 1.0, and 0.1, respectively). Differences between the effects of processing parameters on the inactivation of TI, lectin and urease in soybean were observed and they can be seen either from the mathematical models or the typical figures.The modeling of the effects should help in selection of optimal parameters of hydrothermal treatment for destruction of TI. lectin and urease in soybean. Based on the modeling, lectin and urease can be fully inactivated in soybean treated at proper conditions, while remaining TI activity can be expected in soybean treated at any conditions examined in this study.

Reversal by dithiothreitol of urease inactivation by L-usnic acid

L-Usnic acid inactivates urease through the formation of high M, aggregates of the enzyme. In addition, L-usnic acid strongly binds to the protein, blocking the essential -SH groups of the urease molecule. Low concentrations of dithiothreitol (about 0.8 mM) reverse this blockade without any modification in the pattern of polymerization, inducing the appearance of active high M, polymers.

Reversal by L-cysteine of the inactivation of urease by L-usnic acid

L-usnic acid inactivates urease by inducing the formation of high molecular weight aggregates. L-cysteine reverses this process not by enzyme protection but by stimulating the appearance of active high molecular weight polymers which are inactive under normal conditions.

Shear deformation effects in enzyme catalysis. Metal ion effect in the shear inactivation of urease

The mechanism of the inactivation of the enzyme urease produced by subjecting its dilute solutions to hydrodynamic shear stresses in the range 0.5-2.5 Pa has been determined. By studying the kinetics of urease-catalyzed urea hydrolysis during application of hydrodynamic shear under varying chemical environments, we demonstrate that micromolar quantities of metal ions, in this case adventitious Fe, can accelerate the oxidation of thiol groups on urease and thus inactivate it when the protein is subjected to a shearing stress of order 1.0 Pa. In the absence of metal ion this stress level is ineffectual. It is proposed that this type of synergy between deformation and chemical environment may be crucial in many situations where biological macromolecules are subjected to mechanical stress.

Quaternary structure changes and kinetics of urease inactivation by L‐usnic acid in relation to the regulation of nutrient transfer between lichen symbionts

Abstract L‐usnic acid inactivates urease by the formation of high molecular weight aggregates which can reach a maximum of 700,000 under experimentation conditions previously described. The substrate, urea, itself provokes temporary aggregation states which may be either active or inactive; the latter being reversible. However, these inactive aggregates, of 820,000 molecular weight, are irreversibly stabilized by L‐usnic acid. The active aggregates with molecular weights oscillating from 605,000 to 650,000, depending on whether they are formed in the presence or absence of inactivator, may combine with the substrate to form an apparently normal ES complex.

Stability of urease in soils

Studies with surface samples of Iowa soils selected to obtain a wide range in properties showed that the following treatments of field-moist soils had no effect on urease activity: leaching with water ; drying for 24 h at temperatures ranging from 30 to 60°C ; storage for 6 months at temperatures ranging from −20 to 40°C; incubation under aerobic or waterlogged conditions at 30 or 40°C for 6 months. No loss of urease activity could be detected when field-moist soils were air-dried and stored at 21–23°C for 2yr, but complete loss of urease activity was observed when they were dried at 105°C for 24 h or autoclaved (120°C) for 2h. Inactivation of urease in moist soils was detected at temperatures above 60°C. Treatment of field-moist soils with proteolytic enzymes which cause rapid destruction of jackbean urease did not decrease urease activity, but jackbean urease was destroyed or inactivated when added to sterilized or unsterilized soils. Although no decrease in urease activity could be detected when field-moist soils were air-dried, an appreciable (9–33%) decrease in urease activity was observed when air-dried soils were incubated under aerobic or waterlogged conditions. This decrease occurred within a few days, and prolonged incubation or repetition of the drying-incubation treatment did not lead to a further decrease in urease activity. Treatment of incubated air-dried soil with urease or glucose initially increased urease activity to a level exceeding that of the undried soil, but this activity decreased with time and eventually stabilized at the level observed for the undried soil. The work reported supports the conclusions from previous work that the native urease in Iowa soils is remarkably stable and that different soils have different levels of urease activity determined by the ability of their constituents to protect urease against microbial degradation and other processes leading to inactivation of enzymes.

Enzyme Inactivation by Chlorine Preparations. I. Inactivation of Alcohol Dehydrogenase, Urease, and Trypsin

Enzyme Inactivation by Chlorine Preparations. I. Inactivation of Alcohol Dehydrogenase, Urease, and Trypsin

Heat inactivation of trypsin inhibitor, lipoxygenase and urease in soybeans: effect of acid and base additives.

AbstractEffects of chemical additives on the heat inactivation of trypsin inhibitor (TI), lipoxygenase and urease in soybeans were investigated. The nutritional value of soybeans increases when antigrowth factors, such as TI, are inactivated. Inactivation of lipoxygenase enhances palatability and storage stability. Heat inactivation of antinutritional factors during immersion cooking of dry soymeats was studied without additives. Processing time was varied from 15 min to 2 hr over a temperature range of 120–212 F. The experiments were repeated, with the addition of NaOH or HCl to the cooking water. Without additives, lipoxygenase proved to be the most heat labile and TI, the least. With either acid or base additives, the initial inactivation of urease and lipoxygenase was accelerated significantly; however, while TI inactivation was accelerated by base, it was retarded by acid addition.

Gaseous Loss of Ammonia from Prilled Urea Applied to Slash Pine

AbstractGaseous loss of NH3 from prilled urea, surface applied at 100 kg N/ha to undisturbed dry organic residue over moist soil under slash pine (Pinus elliottii Engelm. var. elliottii), averaged 4% in 7 days as compared with 2% for an area that was control‐burned 5 weeks previous, and 5% for one from which loose debris had been removed. This was approximately equivalent to removal by burning. Residual urea recovery of 41% for the burned area as compared with 16 and 12% for the undisturbed and cleaned areas, indicated urease inactivation and partial soil sterilization by burning. Where urea had been applied to moist undisturbed organic residue, hydrolysis approached completion in 3 days and was accompanied by 9% N loss as NH3. Various types and orientations of needle‐bearing portions of branches of slash pine were submitted to a 75–100 cm drop of prilled urea at 100 kg N/ha. From 0.32–0.87 g of pellets were retained per meter of branch. Aspiration tests under typical alternating night and day humidities and native urease activity showed only 3% loss of this retained urea‐N as NH3 in 4 days. At 8 days an average of 52% of the initially retained N was still dilute‐acid soluble from macerated bark plus needle stubs, with a ratio of seven parts urea N to three parts NH4+‐N. The remainder, except for the small gaseous loss, probably was assimilated.

A semi automated method for plasma arginase

A method for the estimation of plasma arginase is given. It involves preliminary removal of urea with urease, inactivation of urease by phenyl mercuric acetate, followed by incubation of plasma with arginine. The urea produced is estimated on an autoanalyzer. A method for the production of arginase-free urease is given and some factors which influence the results of plasma arginase values are described.

INACTIVATION OF UREASE IN VITRO BY MEANS OF TRITIATED WATER AND ITS RESTORATION WITH POLYCYSTEINE.

INACTIVATION OF UREASE IN VITRO BY MEANS OF TRITIATED WATER AND ITS RESTORATION WITH POLYCYSTEINE.

Inactivation of urease by nitrogen mustard and a comparison with inactivation by x-rays.

SummaryUrease is inactivated by small amounts of nitrogen mustard (Di-2-(chloroethyl) methylamine: HN2) in unbuffered solution at ph 7·0, the inhibition or inactivation being complete when 10−3 M nitrogen mustard is used. With 10−5 M solutions of HN2, 12 per cent inhibition was observed. The inhibition is ph-dependent, the percentage inactivation with 10−3 M HN2 being 100 at ph 6·5 and 0 at ph 5·2. Additions of many different substances protected the urease. Buffer salts protected, citrate and phosphate being most effective; for example, urease was completely protected against inactivation by 10−3 M nitrogen mustard by the addition of 5 × 10−3 M, or stronger, solutions of citrate buffer. Proteins are effective protectors, but nucleic acids are not. All the amino acids tested protect, but cysteine and histidine are much more effective than most others investigated. Blocking of urease —SH groups is also an effective protective measure.The inactivation is considered likely to be a result of the combination o...

Inactivation of urease by x-rays.

SummaryUrease, when irradiated by x-rays in dilute aqueous solutions, is partially inactivated. A dose of 5000 r caused almost complete destruction of the enzyme, and 500 r caused 22 per cent inhibition. Enzyme inactivation was small when measured immediately after the irradiation, but increased gradually during a period of 2 hours after the irradiation. Protection against irradiation could be accomplished in many ways. The enzyme was self-protective, a larger concentration of enzyme reducing the inactivation with a given dose, and other proteins, such as serum albumin, were also effective. Several buffer salts protected, citrate and phosphate being most effective. Amino acids protected, but not to an equal extent, and of those tested, cysteine and methionine were found to be the most, and alanine and tryptophan the least, effective. Addition of versene and the blocking of–SH groups of the enzyme also protected. It is considered likely that the enzyme inactivation is a result of the oxidation of urease —SH groups by free radicals.

The inactivation of urease by deuterons and heat

The inactivation of urease by dry heat indicates that the molecule is not unfolded in the activated state. The inactivation of urease by deuterons shows that the molecular weight of urease is probably in the neighborhood of 100,000, as compared with the previously accepted 480,000.

Interaction of Vitamin C and urease

Vitamin C and Vitamin C-Cu complex inhibit urease activity, the inhibition being very marked in the latter case. The inhibition of urease activity of Vitamin C is not due to dehydro-ascorbic acid as the degree of inhibition produced by it is negligible compared to that produced by Vitamin C at equal concentration. A variety of compounds other than cysteine, like 8-hydroxy-quinoline, sodium-diethyl-dithio-carbamate and potassium cyanide, which are known to protect Vitamin C against oxidation catalysed by Cu++, are shown to protect the urease from inactivation by Vitamin C and Vitamin C-Cu. showing that the inactivation of urease by Vitamin C is related to the oxidation of the vitamin. A well marked correlation between the inactivation of urease on the one hand and the oxidation of Vitamin C on the other has been established. It is suggested that the inactivation of urease by Vitamin C catalyzed by Cu++ may be due to any intermediate products like Cu2O formed during the oxidation.